Pigeon egg white protein-based transparent durable hydrogel via monodisperse ionic surfactant-mediated protein condensation

The thermal gelation property of proteins is useful in creating protein-based materials. The gelation of protein solution often proceeds by the random aggregation of denatured proteins, and the protein-based gels are typically brittle or opaque, or both. Improvement in the mechanical and optical properties of protein-based materials are required for them to be practical and functional. This study investigated pigeon egg white, which is semitransparent in its thermally gelled state, as a protein source for creating hydrogel materials. The protein thermal gelation process was initiated from the orderly condensed state of proteins complexed with monodisperse ionic surfactants to suppress random aggregation. The resultant gel showed transparency in the visible light region and was not destroyed at 99% compression under 17.8 MPa compressive stress, 350-fold higher than the compressive fracture strength of typical boiled pigeon egg white. These results showed that durable transparent hydrogels could be fabricated by the rational combination of natural proteins and surfactants.


Results
Monodisperse surfactants in PC-gel preparation. Boiled pigeon egg white has a softer texture and weak mechanical property on compression than boiled hen egg (Fig. 1b). Hence, we expected that the PCgel prepared from PEW under the same preparation conditions used in previous study would show weaker mechanical properties than HEW PC-gel. The ordering character and arrangement of the constitutive protein of PC can be controlled by the molecular structure of the surfactants and affects the mechanical property of thermally formed PC-gels 11 . In a previous study, we used ionic surfactants prepared from commercially available surfactants with polydisperse PEG chain (Fig. 2a,b). PC is a complex material composed of proteins and surfactants. Since protein is a monodisperse-structured macromolecule, the polydispersity of the surfactants could be a dominant disruption factor in the homogeneity of the protein arrangement inside the PC. Therefore, we used monodisperse surfactants for PC preparation to reduce the heterogeneity of the components in PC for the fabrication of PEW protein-based PC-gel with improved mechanical properties. We synthesized monodisperse anionic and cationic C 12 E 5 surfactants with dodecane chain and pentaethylene glycol chains based on a reported method for monodisperse polyethylene glycols synthesis (Fig. 2c-e) 13,14 . In this study, we used monodisperse ionic C 12 E 5 surfactants and polydisperse ionic C 12 E 4.5 surfactants as used in our previous study (4.5 is the average repeating number of PEG units determined by NMR) 12 to prepare PC and PC-gels and compared their properties to elucidate the effect of the structural homogeneity of the surfactants on the mechanical characteristics of the PC-gels.  www.nature.com/scientificreports/ Preparation and analysis of PCs from PEW solution. We found that PC was formed from the PEW solution by adding a mixture of anionic and cationic C 12 E 5 in an 85/15 ratio (Fig. 3b). In this study, we refer to PC formed using C 12 E 5 surfactants as "PC(PEW-C 12 E 5 )." The PC(PEW-C 12 E 5 ) protein and water contents were 138 mg mL −1 and 71% (w/w), respectively. The constitutive PC(PEW-C 12 E 5 ) proteins were analyzed using SDS-PAGE (Fig. 3c). Ovalabumin (OVA) is the most abundant protein in egg white and exhibits thermal gelation property. HEW contains only one type of OVA, while PEW has two: OVA1 (53.2 kDa, also known as ovalbuminrelated protein Y) and OVA2 (48 kDa) [15][16][17] . SDS-PAGE analysis showed that PC(PEW-C 12 E 5 ) was mainly composed of OVA1 and OVA2. This result supported our idea that PC preparation using PEW as a protein source exhibits thermal gelation properties. The PEW PC with the C 12 E 4.5 surfactant PC(PEW-C 12 E 4.5 ) was also formed by adding a mixture of anionic and cationic C 12 E 4.5 in an 85/15 ratio and showed a protein-based formation yield of 61%. The protein and water contents of PC(PEW-C 12 E 4.5 ) were 137 mg mL −1 and 78% (w/w), respectively.
Thermal gelation of PC(PEW-C 12 E 5 ) and gel transparency. PC(PEW-C 12 E 5 ) was gelled by heating it above 50 °C. We prepared PC(PEW-C 12 E 5 )-gels at 50, 60, 70, 80, and 90 °C. All PC(PEW-C 12 E 5 ) gels were transparent in the visible light region (Fig. 4a,b). PC(PEW-C 12 E 5 )-gel prepared at 70 °C showed the highest transparency and was used in the following experiments. PC(PEW-C 12 E 4.5 ) was also thermally gelled and transparent (Fig. S1). The results that both PC(PEW-C 12 E 4.5 )-gel and PC(PEW-C 12 E 5 )-gel showed the transparent appearance indicates that the optical transparency of the PC-gels is mainly derived from the natural properties of proteins used for PC preparation, rather than from the structure of the surfactant and protein arrangement inside PC that depends on the surfactant structure.
Mechanical properties of the gels. The mechanical strength of PC(PEW-C 12 E 5 ) and PC(PEW-C 12 E 4.5 )gels was measured (Fig. 5). Both PC-gels showed improved mechanical properties compared to the boiled PEW broken at 50 kPa compressive stress (Fig. 1b). Notably, the mechanical properties of PC(PEW-C 12 E 5 )-gel were significantly superior to PC(PEW-C 12 E 4.5 )-gel. PC(PEW-C 12 E 5 )-gel was unbroken under 99% compression at 17.8 MPa compressive stress, while PC(PEW-C 12 E 4.5 )-gel was broken at 2.9 MPa (Fig. 5a-c). The nondestructive and durable property of PC(PEW-C 12 E 5 )-gel in a highly compressed condition were not observed in our previous research on PC gel of HEW prepared using polydisperse surfactants 11 . As shown in the tensile test, PC(PEW-C 12 E 5 )-gel also showed higher deformability and strength than PC(PEW-C 12 E 4.5 )-gel (Fig. 5d). These results supported our idea that the structural homogeneity of the PC state before thermal gelation is the determinate factor in the mechanical properties of the resultant PC-gel and can be improved using monodisperse surfactants Biocompatibility of PC-gel. Cell culture materials are a promising application for hydrogels owing to their water permeability, which is an essential property for the continuous supply of culture media 18 . PC(PEW-C 12 E 5 )-gel showed sufficient transparency for monitoring the culturing process and mechanical strength for future applications in fabricating structural cell culture scaffolds. We tested our PC gel as a cell-culture scaffold material. First, we performed a cytotoxicity assay to investigate the biocompatibility of the gel. HL-60 cells showed no growth under standard culture conditions in the presence of a piece of as-prepared PC(PEW-C 12 E 5 )gel; however, the same assay performed with a piece of boiled PEW showed normal cell growth, suggesting that the ionic surfactants contained in the gel were cytotoxic and inhibited cell growth. Washing the surfactants from the prepared gel was required to safely apply PC gel in cell cultivation systems. Since alcohols and electrolytes inhibit hydrophobic and ionic interactions, respectively, we predicted that the combination of ethanol and PBS could be effective in removing ionic surfactants from PC gels (Fig. 6a). We found that cells can grow in the presence of the gels treated with the mixture of ethanol and PBS; a 40/60 ethanol/PBS ratio was most effective to render PC(PEW-C 12 E 5 )-gel non-cytotoxic. This PC(PEW-C 12 E 5 )-gel washing protocol was also effective for NCI-H460 cell growth (Fig. S2). In compression tests, washed PC(PEW-C 12 E 5 )-gel exhibited mechanical and nondestructive properties similar to the as-prepared gel (Fig. S3).
Cell growth on PC-gel. Finally, we investigated the morphology of cells grown on PC gel. NCI-H460 cells were cultured on planar PC(PEW-C 12 E 5 )-gel and in a standard dish as a control. The control cells adhered to and propagated on the dish surface (Fig. 6b,c); whereas, the cells on planar PC(PEW-C 12 E 5 )-gel as a substrate supporting material formed living cell aggregates that did not adhere to the gel (Fig. 6d,e). The non-adhesiveness, improved strength, and transparency of PC gel could be advantageous in biocompatible structured scaffold material fabrication for three-dimensional cell culture used in preparing spherical, multicellular aggregates (spheroids) which could be utilized for biological application such as tissue engineering, developing of Organson-a-Chip, and as a model system for cell self-organization and metabolism research 19 .

Discussion
This study was inspired by the transparency of thermally gelled PEW (boiled egg state). Although the molecularlevel origin of the transparency has not yet been investigated, PEW proteins have been used as a protein source in creating hydrogels with improved optical transparency. It was shown that thermally treated PEW protein at PC state formed with newly synthesized monodisperse surfactants resulted in a transparent durable hydrogel with highly improved mechanical properties that showed nondestructive characteristics under 99% compression. Compared with the results of polydisperse surfactants, these results revealed that the structural homogeneity of the surfactant was an important determining factor in the PC gel's mechanical properties. After washing to remove cytotoxic surfactants, PC gel could be used for biotechnological applications such as scaffold materials in cell cultivation systems. This study established a novel method for creating transparent, durable protein-based gel materials from natural protein sources using monodisperse ionic surfactants. Understanding the molecular mechanism of the origin of the optical transparency of egg white proteins in the thermal gelled state is important for the future application of egg white proteins. Since egg white is a complex system containing various proteins, www.nature.com/scientificreports/ it is difficult to analyze the physical properties of egg white in solution and in the gelled state. Detailed analysis of the structure-functional properties of egg white proteins in the standard solution and PC states, and their gel states, using purified egg white proteins is necessary for future research. Also, the structural analysis in this study is insufficient to reveal the high-strength nature of PC gels. In the research stage of PC gels using purified proteins, detailed properties of our protein condensate gel could be characterized by various light scattering methods, which are effective for gel structure analysis, and by direct structural observation using electron microscopy and AFM. The properties of protein condensate gel will be better understood through such structural analysis, leading to the developing of novel biomaterials using natural protein resources in the future.

Methods
Preparation of pigeon egg white (PEW) solution. PEW was collected from fresh market-purchased pigeon eggs, filtered twice through a mesh, diluted with an equal weight of water, stirred for 30 min at 4 °C to homogenize the solution, and centrifuged (10,000g for 10 min). The supernatant was dialyzed twice using a 1000 MW cutoff dialysis bag against water for 12 h to remove small molecular substances and centrifuged (10,000g for 10 min) to remove insoluble aggregates. The clear homogenized supernatant was stored at 4 °C. Protein concentration of the PEW solution was measured by BCA method (BCA Protein Assay Kit, Thermo Fisher Scientific, USA) with bovine serum albumin as the standard.

Synthesis and characterization of surfactants. Polydisperse anionic C 12 E 4.5 was purchased from
Sigma-Aldrich (catalog 463256) and purified, polydisperse cationic C 12 E 4.5 was synthesized from polydisperse anionic as previously reported 12 . Monodisperse anionic and cationic C 12 E 5 surfactants were newly synthesized in this study (the detail of the synthesis are described in supplemental information). ES-TOF MS analyses of surfactants were performed with a Q-TOF Micro spectrometer (Waters Corp., USA) in a positive mode.

Preparation of surfactant solution.
Anionic and cationic surfactants were dissolved in MilliQ-grade water. The solutions were neutralized with NaOH and HCl, respectively, and adjusted to 100 mM. The surfactant solutions were stored at room temperature.

Preparation of PC(PEW-C 12 E 5 ) and PC(PEW-C 12 E 4.5 ).
Solutions of 100 mM anionic and cationic C 12 E 5 surfactants were mixed in a volume ratio of 15/85, and 100 μL of the mixed solution was added to 1 mL of 10 mg mL −1 PEW solution. The mixed solution was centrifuged (10,000g for 2 min), and PC(PEW-C 12 E 5 ) was observed at the bottom of the tube as a transparent liquid. The procedure can be scaled up to prepare large quantities. PC(PEW-C 12 E 4.5 ) was obtained by adding 80 μL of 100 mM mixture of anionic and cationic C 12 E 4.5 surfactants (anionic/cationic = 15/85) to 1 mL of 10 mg mL −1 PEW solution. The PC formation yield was calculated from the protein concentration (measured by BCA method) of the supernatant after PC formation as follows: Formation yield (%) = 100 × 1 − protein sup / protein 0 ,  Preparation of PC-gels. PC(PEW-C 12 E 5 ) and PC(PEW-C 12 E 4.5 ) were poured into appropriate molds and heated at 50, 60, 70, 80, and 90 °C for 20 min in a water bath or by sandwiching between heat blocks. The prepared gels were stored in water at 4 °C.

Measurement of protein content of PC.
An aliquot of PC(PEW-C 12 E 5 ) and PC(PEW-C 12 E 4.5 ) was mixed with a ninefold volume of buffer (20 mM Tris-HCl, pH 8.0, containing 200 mM NaCl). Under these electrolyte conditions, the PCs were reconverted to an aqueous protein solution. The protein concentration of the solution was measured by BCA method with bovine serum albumin as the standard. The result was multiplied by ten to determine the protein content of PC.
Measurement of water content of PC. The PC water content was measured as the difference between the mass weight of the wet and dry samples. The dried samples were prepared in a vacuum at 70 °C for 6 h. PC gel cytotoxicity evaluation using HL-60 cells. Human promyelocytic leukemia cells (HL-60) (iCell Bioscience Inc., Shanghai, China) in Iscove's Modified Dulbecco's Medium (IMDM) and 20% fetal bovine serum (FBS) medium were cultured in a CO 2 incubator at 37 °C with 5% CO 2 . Two milliliters of 5 × 10 5 cells mL −1 culture medium cell density were added to 3.5 cm diameter Petri dishes and cultured at 37 °C with 5% CO 2 for 48 h in the presence of a 20 mm × 15 mm × 1 mm piece of washed gel. One hundred microliters of the culture were collected every 12 h, mixed with 10 μL of CCK-8 reagent (Cell Counting Kit-8, Dojindo, Inc., Kumamoto, Japan), incubated for 3 h in a CO 2 incubator, and the absorbance at 450 nm was measured. All experiments were performed in triplicate and averaged. The same experiment was performed in the absence of a piece of the gel as a positive control group.

Measurement of transparency of PC-gels.
Cell culture on gel surface. Cell growth on the surface of the planer-shaped PC(PEW-C 12 E 5 )-gel was observed. The planar-shaped PC(PEW-C 12 E 5 )-gel (20 × 15 × 1 mm) was washed as described above using a 40/60 ethanol/PBS ratio. The washed gel was placed in a 3.5 cm Petri dish. Three milliliters of NCI-H460 cells suspended in RPMI 1640 and 10% FBS medium 2 × 10 5 cells mL −1 density was seeded in the dish and cultured in a CO 2 incubator at 37 °C with 5% CO 2 for 96 h. The culture medium was replaced with fresh medium at the time of 48 h. The same experiment was performed without the gels, and cells were cultured on a tissue culture-treated culture dish (Corning Inc., #430165). Cells were observed at 48 and 96 h using a microscope (Eclipse Ti2-U, Nikon, Japan, CCD camera DS-Ri2, Nikon, Japan). 96 h-cultivation cells were stained with -Cellstain-Double Staining Kit (Dojindo, Inc., Kumamoto, Japan), and the living cells were observed using a fluorescent microscope (Eclipse Ti2-U, Nikon, Japan, CCD camera DS-Ri2, Nikon, Japan).

Data availability
All data generated or analyzed during this study are included in this published article (and its supplementary information files).